The invention is in the field of genetic engineering by recombinant DNA technology, more particularly the genetic engineering of potato plants in order to change the starch composition in the tubers towards essentially amylose-free starch.
Starch is the major storage carbohydrate in potato and consists of two components, a linear (1xe2x86x924)xcex1-D-glucan polymer and a branched (1xe2x86x924) (1xe2x86x926)xcex1-D-glucan called amylose and amylopectin, respectively. Amylose has a helical conformation with a molecular weight of 104-105. Amylopectin consists of short chains of xcex1-D-glucopyranose units primarily linked by (1xe2x86x924)xcex1 bonds with (1xe2x86x926)xcex1 branches and with a molecular weight up to 107. In plants starch is found in two types of plastids: chloroplasts and amyloplasts. In both types of organelles the starch occurs as granules. In chloroplasts so-called transitory starch is accumulated for only a short period of time, whereas starch in amyloplasts is accumulated for long term storage and hence is named reserve starch. Generally, amylose makes up 11%-37% of the total reserve starch and variation in the amylose content is not only found among different plant species, but also among different cultivars of the same species. In potato the amylose content in the tuber varies from 18% to 23%. Furthermore, in a number of plant species mutants are known with a starch composition which deviate significantly from the above mentioned percentages.
Transitory and reserve starch are generally considered to be synthesized by the same enzymes. Starch metabolism in leaves follows a diurnal rhythm: synthesis and accumulation occur during the light period while hydrolysis occurs during the night. In storage tissue, starch synthesis occurs during a specific phase of tissue development; the synthesis being the predominant function of amyloplasts. The amount of amylose found in storage tissue of potato is about twice as high as that in leaves.
Sucrose is considered to be the major substrate for starch biosynthesis which involves the following steps: initiation, elongation, branching and granule formation. In the pathway of conversion of sucrose into amylose and amylopectin at least 13 enzymes play a role. Three groups of enzymes are directly involved in the formation of starch. These enzymes are phosphorylase, starch synthases and branching enzymes. Phosphorylase is active in starch breakdown, branching enzyme converts amylose into amylopectin by the breakage of (1xe2x86x924)xcex1-bonds and the synthesis of (1xe2x86x926)xcex1-bonds. Starch synthases are responsible for the synthesis of starch by the addition of ADP (UDP) glucose subunits to the non-reducing end of an (1xe2x86x924)xcex1-D-glucan polymer. Starch synthase has been identified in two forms: one form is soluble while the other is tightly associated with starch granules. The soluble enzyme uses only ADP-glucose as the D-glucosyl donor, whereas the granule bound starch synthase (GBSS) utilizes ADP-glucose and UDP-glucose. Solubilization of the GBSS protein from starch granules of various plants has been reported. Although in maize there are thought to be at least two forms of GBSS, potato seems to have only one form. The presence and activities of the different starch synthases are important to starch biosynthesis not only because they have an effect on the amylose/amylopectin ratio in starch, but also because they can have a large impact on total starch content. In general, it appears that complete suppression of the enzymes producing amylose can be achieved with almost no change in the total amount of starch laid down, whereas suppression of the enzyme system producing amylopectin leads to a marked decrease of the amount of total starch.
Starch as such or in modified form is widely used in the food, paper and textile industries. With the depletion of natural oil resources starch could also become a substitute for oil as a raw material for the chemical industry. Therefore, it could become of major interest to produce starch which meets special requirements for certain applications. Although special forms of starch are already available from mutants of maize and rice and starches from other sources might have certain advantages, genetical engineering could be an option in order to obtain tailor-made starches in plants in which (recessive) mutants are not easily obtained. Selection of mutants is especially difficult in vegetatively propagated crops which are mainly crosspollinators and/or polyploids, such as the potato.
Although recently in a laborious isolation procedure a mutant of potato (amf) was isolated which, in analogy to the wx mutants in maize, lacks GBSS protein, GBSS activity and amylose (Hovenkamp-Hermelink et al. 1987), the breeding of such a mutant into a cultivar will take another number of years. One cause for the long duration of the procedure is the fact that a haploid clone had to be used for the isolation of the recessive mutant. To circumvent problems of isolating recessively inherited mutants in a polyploid crop like potato and to speed up the introduction of such a mutant character in potato varieties, the antisense approach would be a very important alternative, because an antisense gene would act as a dominant suppressor gene. The great advantage is that eventually it will become possible to mimick such a mutant phenotype directly in a tetraploid variety. With the availability of GBSS sequences, both from maize (Shure et al 1983) and potato (Hergersberg 1988; Visser et al 1989d) and an efficient transformation system for potato (Visser et al 1989a, 1989b) this approach could be tested.
It has been shown that antisense RNA transcripts can be used to mimic mutations in pro- and eukaryotes (for review see van der Krol et al. 1989). Antisense RNA was originally found as a naturally occurring mechanism used to control gene expression in bacteria (Tomizawa et al. 1981; Mizuno et al. 1984). Izant and Weintraub (1984, 1985) proposed that antisense RNA could be used to inhibit the expression of eukaryotic genes. By inhibiting the expression of specific target RNAS, this approach has led to the generation of mutant phenotypes in a number of different eukaryotic systems. In plants the use of antisense RNA proved to be successful in effectively inhibiting the activity of nopaline synthase (Rothstein et al. 1987; Sandler et al. 1988), chloramphenicol acetyltransferase (Ecker and Davis 1986; Delauney et al. 1988), chalcone synthase (van der Krol et al. 1988), polygalacturonase (Smith et al. 1988; Sheehy et al. 1988), phosphinotricin acetyl transferase (Cornelissen and Van de Wiele 1989) and xcex2-glucuronidase (Robert et al. 1989).
Visser (1989) tested whether the antisense approach could be used to inhibit the expression of the gene for granule-bound starch synthase in potato using heterologous antisense constructs, i.e. an antisense gene constructed from a maize genomic GBSS gene.
The antisense gene was fused between the 35S cauliflower mosaic virus promoter and the nopaline synthase terminator in the binary vector pROK-1, which also carries a plant selectable kanamycin resistance gene. Since it was known from the amf-mutant that the mutation is expressed in all tissues in which starch is formed, including columella cells of the root cap, it was expected that also antisense effects would be visible in roots. The presence or absence of amylose could be easily detected because amylose forms a blue staining complex with the iodine present in Lugol""s solution (Ixe2x80x94KI). Starch without amylose, i.e. only containing amylopectin, forms a reddish-brown staining complex with iodine. In order to efficiently test the introduced antisense gene in potato for a biological effect a transformation system was developed in which the binary antisense vector was incorporated into Agrobacterium rhizogenes. The binary vector was present next to the wildtype Ri-plasmid of A. rhizogenes which is responsible for the formation of so-called hairy roots on plant explants. Agrobacterium rhizogenes was used instead of Agrobacterium tumefaciens because it is possible to screen for an effect of the introduced constructs already after 10 days by staining hairy roots with Lugol""s solution and because plants can be easily regenerated from hairy roots. In this way heterologous (maize) binary antisense GBSS plasmids were transferred by A. rhizogenes to stem segments from potato.
Both in untransformed or otherwise transformed wildtype roottips never anything else than blue staining roottips were present. Hairy roots obtained after transformation with A. rhizogenes carrying heterologous binary antisense GBSS plasmids were analyzed for the presence or absence of amylose in their starch by staining the roottips with Lugol""s solution. The majority of the roots stained blue as wildtype untransformed roots did. However, some roots (1-15% of the stained roots) had a color pattern different from that of wildtype roots in that the central cells of the root cap were blue and the cells towards the outside of the rootcap were red. These intermediate colouring roots were indications that the inserted antisense genes had some effect on the amylose content. Root clones were established and subcultured and roottips were investigated every fortnight during six weeks of culture. The results of these experiments showed that instability of color patterns occurred at a rather high frequency. The instability of the effect in columella cells was the reason to regenerate plants from kanamycin resistant hairy roots irrespective of their color.
On plants, regenerated from kanamycin resistant hairy roots, microtubers as well as soil grown tubers were induced. Analysis of these tubers showed that none of them had red or intermediate staining starch. All tubers showed blue staining (=amylose containing) starch. Starch isolated from these tubers was analyzed for the presence of GBSS protein and GBSS activity and for the presence of amylose. In all tubers tested GBSS protein was, seemingly unaltered, present. However, GBSS activity in particular and to a much lesser degree amylose content were affected in starch preparations from a number of transformed plants. As shown in FIG. 2A, the untransformed wildtype (PD007) and a pBI121 transformed wildtype (Ri-007) had similar GBSS activities, while the amf-mutant had no detectable GBSS activity. GBSS activity was inhibited significantly in the antisense GBSS transformants down to only 10% of that found in wildtype plants. Total inhibition of GBSS activity was not obtained in any of the transformants analyzed. The amylose content measurements gave a different picture. Although in almost all cases there was a somewhat lower amylose content, the difference was significant in only two cases (R-196 and R-227, FIG. 2B). The maximum reduction of the amylose content was found in transformant R-196, which also had the lowest GBSS-activity; a reduction down to 78% of the wildtype amylose content. Molecular analyses of the antisense transformants revealed that the number of integrated antisense copies was 1 to 4, but only those plants which contained three or more copies of the antisense GBSS construct showed a pronounced effect on GBSS activity. It is evident from these observations that the effect of a lower GBSS activity on the amylose/amylopectin ratio is not straightforward.
The results described resemble very closely the situation obtained in tomato when using antisense poly-galacturonase genes. A reduction of 90% of the polygalacturonase activity does not have a great effect on the lycopene content (Sheehy et al. 1988, Smith et al. 1988).
The above results were not too encouraging, but it was nevertheless decided to expand the investigations to homologous constructs derived from a full-length potato GBSS cDNA.
Surprisingly, it was found that it is possible to inhibit the expression of granule-bound starch synthase (GBSS) in potato, and thus affect the amylose content of potato tuber starch, by stably introducing homologous antisense constructs. The results described show that it is possible using the antisense approach to interfere with enzymes in biosynthetic pathways such as starch biosynthesis. In using this technique loss of function mutations, such as the amf mutation, which are principally inherited recessively can be mimicked, because antisense genes act as dominant (hemizygous) genes suppressing translation of mRNA.
Surprisingly, it was subsequently found that the effect of essentially amylose-free tuber starch could also be obtained by stably introducing homologous sense constructs, e.g. based on potato GBSS genomic DNA. A phenomenon known as co-suppression appears to occur; it is not yet possible to give an explanation of it.
The invention provides a potato plant which has a genome containing, as a result of genetic engineering, at least one gene construct containing a potato granule-bound starch synthase (PGBSS) cDNA or genomic DNA sequence in reverse or functional orientation in an expression cassette which is functional in potato plants, said gene construct giving rise to tubers containing essentially amylose-free starch.
In one preferred embodiment, said gene construct contains a PGBSS cDNA sequence in reverse orientation which results in the production of PGBSS antisense RNA.
In another preferred embodiment, said gene construct contains a PGBSS genomic DNA sequence in functional orientation which results in co-suppression of PGBSS enzyme activity.
The invention further provides cells, parts and tubers of said potato plant, and essentially amylose-free starch from it.
The invention will be illustrated by means of examples which are given for illustrative purposes only and may not be construed as limiting the scope of the invention. For example, the transformation system used in example 1 (Agrobacterium rhizogenes) may be replaced by any suitable alternative, such as the Agrobacterium tumefaciens transformation system (see ex. 2) or the direct gene transfer technique (DGT). Such alternatives are well known to the person skilled in the art. A survey of transformation systems suitable for potato is given in chapter I of Visser (1989).
Similar remarks apply to the choice of the transformation vector (if any), the elements of the expression cassette, the selection markers, etc. For example, the PGBSS promoter may be used to regulate the transcription of the sense or anti-sense PGBSS DNA, instead of the CaMV promoter used in example 1. The sense or anti-sense PGBSS cDNA or genomic DNA sequence does not have to cover the complete coding sequence but should cover a sufficient part of it to be effective for obtaining tubers containing essentially amylose-free starch. At present, the use of anti-sense PGBSS cDNA is preferred above using anti-sense PGBSS genomic DNA. The gene construct used may contain the PGBSS DNA (preferably genomic DNA) in its functional orientation and yet result in essentially amylose-free tuber starch.